N-acetyl mannosammine as a therapeutic agent

ABSTRACT

The invention relates to compositions and methods for treating kidney and muscle dysfunction that involves use of therapeutic amounts of N-acetyl mannosamine.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 15/702,529,filed on Sep. 12, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/754,304, filed Jun. 29, 2015, now abandoned,which is a continuation of U.S. patent application Ser. No. 13/791,576,filed Mar. 8, 2013, and issued as U.S. Pat. No. 9,095,597 on Aug. 4,2015, which is a continuation of U.S. patent application Ser. No.12/530,433, filed Mar. 19, 2010, and issued as U.S. Pat. No. 8,410,063on Apr. 2, 2013, which is the U.S. National Stage of InternationalApplication No. PCT/US2008/006895, filed May 30, 2008, which waspublished in English under PCT Article 21(2), which in turn claims thebenefit of U.S. Provisional Patent Application No. 60/932,451, filed May31, 2007. The prior applications are all incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant no. ZIAHG000215 and grant no. ZIA HG200322 awarded by the National Institutesof Health, National Human Genome Research Institute; the government hascertain rights in the invention.

GOVERNMENT FUNDING

The invention described herein was developed with support from theNational Human Genome Research Institute (NHGRI), which is part of theNational Institutes of Health (NIH). The United States Government hascertain rights in the invention.

FIELD OF THE INVENTION

The invention is related to a methods and compositions involving use ofthe neutral sugar N-acetyl mannosamine (ManNAc) for therapeutic purposesin humans. Such therapeutic uses include treatment of myopathies (e.g.,hereditary inclusion body myopathy (HIBM)) and certain kidney diseases(e.g., those involving proteinuria and hematuria).

BACKGROUND OF THE INVENTION

Hereditary inclusion body myopathy (HIBM; OMIM 600737) is a rareautosomal recessive neuromuscular disorder. Argov, et al., Neurology 60,1519-1523 (2003); Eisenberg, et al. (2001) Nat Genet 29, 83-87 (2001);Griggs, et al. (1995) Ann Neurol 38, 705-713 (1995). The disease usuallymanifests at approximately 20 years of age with foot drop and slowlyprogressive muscle weakness and atrophy. Histologically, it isassociated with muscle fiber degeneration and formation of vacuolescontaining 15-18 nm tubulofilaments that immunoreact like β-amyloid,ubiquitin, prion protein and other amyloid-related proteins. Askanas etal. Curr Opin Rheumatol 10, 530-542 (1998); Nishino, et al. (2005) ActaMyol 24, 80-83 (2005); Askanas, et al. Ann Neurol 34, 551-560 (1993);Argov, et al. Curr Opin Rheumatol 10, 543-547 (1998). Both weakness andhistological changes initially spare the quadriceps. However, thedisease is relentlessly progressive, with patients becomingincapacitated and wheelchair-confined within two to three decades. Thereis no treatment available.

Accordingly, new compositions and methods are needed for treatinghereditary inclusion body myopathy and related diseases.

SUMMARY OF THE INVENTION

One aspect of the invention is a therapeutic method for increasingsialic acid in a mammal in need thereof comprising administering to themammal an effective amount of N-acetyl mannosamine or a derivativethereof to thereby increase sialic acid in the mammal.

In some embodiments, such methods are performed to treat a disease orcondition. For example, such a disease can be muscular atrophy or kidneydisease. In general, the types of muscular atrophies that can be treatedby the methods and compositions of the invention are those caused bysialic acid deficiency. Examples of such muscular atrophy diseases andconditions include distal myopathy with rimmed vacuoles (Nonakamyopathy) and hereditary inclusion body myopathy.

Surprisingly, the compositions and methods of the invention are usefulfor treating certain kidney conditions and diseases, for example, thoseinvolving proteinuria, hematuria resulting primarily or secondarily fromhyposialylation (lack of sialic acid). Thus, the present methods areeffective for treatment of kidney disorders due to poor kidney membraneformation and/or function. For example, kidney membranes that areaffected by loss of sialic acid include the glomerular basement membraneand/or the podocyte membranes. Hence, the invention is useful fortreating malformed or poorly functioning glomerular basement membranesand/or podocyte membranes. In general, the methods of the invention canincrease sialylation of kidney podocalyxin, improve podocyte footprocess morphology and/or improve glomerular basement membraneintegrity.

Another aspect of the invention is a method of treating a kidneydisorder in a mammal comprising administering a therapeutic amount ofN-acetyl mannosamine or a derivative thereof to the mammal, wherein thekidney disorder involves proteinuria and hematuria. For example, such atherapeutic amount of N-acetyl mannosamine or a derivative thereof isabout 1 g to about 20 g per day.

DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates the intracellular metabolism of sialicacid. This figure shows portions of a cell, where the nucleus isdepicted at the bottom (below the depicted nuclear membrane) and thecytoplasm is at the top of the figure. Cytosolic glucose is converted inseveral steps into UDP-GlcNAc, which serves as substrate for thebi-functional, rate-limiting, committed enzyme for sialic acidbiosynthesis: UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE/MNK). GNEcatalytic activity (EC 5.1.3.14) epimerizes UDP-GlcNAc to ManNAc,followed by the phosphorylation of ManNAc to ManNAc-6-phosphate(MaNAc-6-P) by the MNK kinase catalytic domain (EC 2.7.1.60).ManNAc-6-phosphate is then condensed with phosphoenolpyruvate toNeu5Ac-9-phosphate by Neu5Ac-9-P synthetase. The phosphate is thenreleased and Neu5Ac is activated by CMP-Neu5Ac synthetase to CMP-Neu5Acin the nucleus. CMP-Neu5Ac then enters the trans-Golgi and serves as thesubstrate for different sialyltransferases involved in the production ofsialylated glycoconjugates. These are subsequently cleaved in thelysosome to yield free sialic acid, which is exported to the cytoplasmand re-utilized or degraded to ManNAc and pyruvate. CMP-sialic acidstrongly feedback inhibits the GNE epimerase at its allosteric site.

FIG. 2A-E illustrates the generation and identification ofGneM712T/M712T knockin mice. FIG. 2A is a schematic diagram illustratingthe murine Gne (Ueal) genomic locus, exons 11 and 12, after homologousrecombination with the sequence-verified targeting vector. The M712Tmutation was created in exon 12, and a neo cassette (under the PGKpromoter) flanked by flippase recombinase target (FRT) sites wasinserted. LoxP sites were inserted before exon 12 and after the PGK-neogene. FIG. 2B shows the genotyping of mutant mice. A PCR-amplified387-bp fragment of genomic DNA across the M712T (ATG to ACG) mutationwas digested by the NlaII restriction endonuclease into 265-bp, 89-bp,and 33-bp fragments in a wild-type allele (+) and into 354-bp and 33-bpfragments in a mutant M712T allele (−). MW, molecular weight. FIG. 2Cshows the results of RT-PCR of kidney and skeletal muscle RNA. RNA wasreverse transcribed and PCR-amplified using primers covering exons 11and 12 (355 bp). Digestion by NlaIII cut the wild-type allele (+) into225-bp, 89-bp, and 41-bp fragments and the mutant M712T allele (−) into314-bp and 41-bp fragments. Digestion confirmed the exclusive presenceof the mutant M712T allele in Gne^(M712T/M712T) (−/−) tissues. FIG. 2Dshows the numbers and genotypes of mice at E17-E19 and P21. At P21,genotyping of 76 mice from 13 litters (9 GneM^(712T/+) matings)identified only 1 GneM^(712T/M712T) offspring. Subsequent genotyping of35 E17-E19 embryos from 4 Gne^(M712T/+) mice yielded a Mendeliandistribution of genotypes. FIG. 2E shows that at P2, Gne^(M712T/M712T)pups were smaller than their heterozygous (GneM^(712T/+)) and wild-type(Gne^(+/+)) littermates and lacked a prominent milkspot.

FIG. 3A-E provides results of histological kidney analyses. FIG. 3Aillustrates gross kidney pathology. Kidneys of Gne^(M712T/M712T) miceshowed hemorrhages but were normal in size and shape compared withkidneys of wild-type (Gne^(+/+)) and heterozygous (GneM^(712T/+))littermates. FIG. 3B shows representative H&E-stained sections of renalcortex (c) and medulla (m) showing tubular dilatations inGne^(M712T/M712T) mice (arrows). Scale bars: 1,000 μm. FIG. 3C provideshigh magnification images of collecting ducts, renal tubules, andurinary space, filled with red blood cells in Gne^(M712T/M712T) mice.Scale bars: 100 μm. FIG. 3D provides high magnification images ofglomeruli (g) with red blood cells infiltrated into the Bowman space inGne^(M712T/M712T) mice. Scale bars: 100 μm. FIG. 3E shows representativesections of normal glomerulus (DICII, left panel) demonstrating theabundance of Gne/Mnk protein inside the glomerular space uponimmunolabeling with Gne/Mnk antibodies (FITC filter, right panel). Scalebars: 50 μm.

FIG. 4A-D shows transmission electron microscope images of mouse kidneysections. FIG. 4A shows representative cross-sections of glomerularcapillaries in the juxtamedullar zone of a wild-type mouse (age P2).Enlarged insets (right panels) show detailed endothelial cells (ec),glomerular basement membrane (GBM); arrowheads), and foot processes (fp)of the glomerular epithelial cells (podocytes) with well formed, openfiltration slits (asterisks). FIG. 4B shows representativejuxtamedullary glomerular capillaries of a Gne^(M712T/M712T) mouse (ageP2, littermate of the wild-type mouse shown in FIG. 4A), indicatingsegmental splitting of the lamina densa of the GBM (arrowheads) as wellas dramatically flattened and fused podocyte foot processes lining theGBM. The filtration slits are sparse and irregular in shape andposition. Insets (right panels) show fused foot process membranes andformation of abnormal tight junction-like structures at the filtrationslits (diamonds). FIGS. 4C and 4D representative glomerular capillariesof a Gne^(M712T/M712T) mouse following ManNAc treatment (age P19). Theintegrity of the GBM as well as the formation of podocyte foot processesand the number of filtration slits were all improved when compared withthe untreated Gne^(M712T/M712T) mouse in FIG. 4B. Some filtration slitswere open, while others still formed tight junctions. The GMB showedoccasional small stretches of areas where splitting was apparent(arrowheads). L, capillary lumen; N, nucleus; us, urinary space. Scalebars: 1 μm.

FIG. 5A-5F illustrates the biochemistry and renal histology of knockinmice following ManNAc treatment. FIG. 5A shows the numbers of micesurviving past age P3 after ManNAc administration in the drinking waterof Gne^(M712T/+) mice. Six Gne^(M712T/+) mice received 1 mg/ml (˜0.2g/kg/d) ManNAc; 7 total litters were scored; 13 pups died at age P1-P3.Seven Gne^(M712T/+) mice received 5 mg/ml (˜1 g/kg/d) ManNAc; 13 totallitters were scored; 14 homozygous mutant pups died at age P1-P3. Thepercentage of survivors of each genotype is indicated. FIG. 5B-5D showsrepresentative H&E-stained kidney sections showing renal cortex andmedulla (FIG. 5B); collecting ducts, renal tubules, and urinary space(FIG. 5C); and glomeruli (FIG. 5D) following ManNAc feeding at age P6.Wild-type (Gne+/+) kidneys showed normal histology. Gne^(M712T/M712T)kidneys showed a range from very mild (middle panel) to moderatelysevere (right panel) red blood cell infiltrations, but in all cases lesssevere than in untreated Gne^(M712T/M712T) mice at age P2 (FIG. 5E-G).Scale bars: 500 μm (FIG. 5B), 100 mm (FIGS. 5C and 5D). FIG. 5E showstwo ManNAc-treated (˜1 g/kg/d) 6-week-old male littermates. Survivinghomozygous mutant mice (Gne^(M72T/M712T)) were smaller than theirwild-type littermates. FIG. 5F shows Gne/Mnk epimerase enzymaticactivities in skeletal muscle. Administration of ManNAc (shaded bars)increased the activity in wild-type muscle from 100% to 114% (±19.7)(n=3; P=0.2) and increased the activity in homozygous mutant(Gne^(M712T/M712T)) muscle from 19.4% (±7.5) to 31% (±8.4) (n=7;P=0.05).

FIG. 6A-E shows immunoblots of muscle, kidney, and brain extracts ofknockin mice. Immunoblots of muscle (FIG. 6A) and kidney (FIG. 6B)extracts exhibited decreased Gne/Mnk protein expression (upper band,arrows, 79 kDa) in homozygous mutant Gne^(M712T/M712T) (−/−) micecompared with heterozygous (+/−) and wild-type (+/+) littermates(normalized to β-actin). Gne/Mnk protein expression increased uponManNAc feeding in Gne^(M712T/M712T)(−/−) tissues when compared withuntreated tissues. FIG. 6C shows immunoblots of kidney extracts labeledwith laminin-1 antibodies. No difference in laminin-1 intensity wasdetected (n=6; P=0.65) between Gne^(+/+) (+/+) and GneM^(712T/M712T)(−/−) littermates without or with ManNAc treatment. FIG. 6D showsrepresentative immunoblots of brain extracts labeled with PSA-NCAMantibodies. Upon ManNAc treatment, the intensity of the PSA-NCAMsignals, reflecting sialylation status, increased by 2% to 28% intreated Gne^(M712T/M712T) (−/−) brain (n=14) when compared withuntreated brain (n=10). FIG. 6E shows immunoblots of kidney extracts(age P2) labeled with antibodies against podocalyxin (˜140-150 kDa).Top: Following desialylation of Gne^(+/+) (+/+), Gne^(M712T/+) (+/−), orGne^(M712T/M712T) (−/−) kidney extracts by neuraminidase (lanes 2 and4), podocalyxin migrated more slowly (˜160-180 kDa) than untreatedsamples (lanes 1 and 3). Gne^(M712T/M712T)(−/−) kidney extracts (lanes 5and 6) contained desialylated podocalyxin. Bottom: Sialylation ofpodocalyxin at P6 in Gne^(M712T/M712T)(−/−) mice changed significantlyafter ManNAc treatment (lanes 3 and 4).

FIG. 7 schematically illustrates a timeline for administration ofN-acetyl mannosamine during a clinical trial of human patients.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file4239-83432-17_Sequence_Listing.txt, May 14, 2019, 16.5 KB], which isincorporated by reference herein.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, N-acetyl-mannosamine and derivatives thereofare useful for treating a variety of diseases and conditions.N-acetyl-D-mannosamine is a key compound in the sialic acid biosyntheticpathway (see FIG. 1). In particular, there is a regulated, rate-limitingenzymatic step in the pathway that leads to sialic acid formation, andthis rate-limiting step gives rise to N-acetyl-D-mannosamine. Hence,once N-acetyl-D-mannosamine is formed or administered, no otherenzymatic step leading to the formation of sialic acid is subject tofeedback inhibition. Thus, administration of N-acetyl-D-mannosamine willlead to increased amounts of sialic acid. The structure ofN-acetyl-mannosamine is shown below.

Therefore, according to the invention, administration of N-acetylmannosamine (ManNAc) and/or its derivatives promotes formation of sialicacid (N-acetylneuramic acid). Sialic acids are sugars found on manycellular and tissue components. For example, sialic acids are present onmost cell surfaces, and on proteins and lipids and are involved in cellto cell interactions. Sialic acid-rich oligosaccharides on theglycoconjugates found on surface membranes help keep water at thesurface of cells. The sialic acid-rich regions also contribute tocreating a negative charge on the cells surface. Since water is a polarmolecule, it is attracted to cell surfaces and membranes. Thus, sialicacids contribute to cellular hydration and fluid uptake. Sialic acid isalso a vital component of many body fluids including, serum,cerebrospinal, saliva, amniotic, and mother's milk.

N-Acetylmannosamine Derivatives

According to the invention, N-acetylmannosamine and derivatives thereofcan also be used in the therapeutic methods and compositions of theinvention. The structures of such N-acetylmannosamine derivatives usefulin the invention are defined by Formula I.

wherein:

R₁, R₃, R₄, or R₅ is hydrogen, lower alkanoyl, carboxylate or loweralkyl; and

R₂ is lower alkyl, lower alkanoylalkyl, lower alkyl alkanoyloxy.

The following definitions are used, unless otherwise described: Alkyl,alkoxy, alkenyl, alkynyl, etc. denote both straight and branched groups;but reference to an individual radical such as “propyl” embraces onlythe straight chain radical, a branched chain isomer such as “isopropyl”being specifically referred to.

Lower alkyl refers to (C₁-C₆)alkyl. Such a lower alkyl or (C₁-C₆)alkylcan be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl,pentyl, 3-pentyl, or hexyl; (C₃-C₆)cycloalkyl can be cyclopropyl,cyclobutyl, cyclopentyl, or cyclohexyl; (C₃-C₆)cycloalkyl(C₁-C₆)alkylcan be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl,2-cyclopentylethyl, or 2-cyclohexylethyl; (C₁-C₆)alkoxy can be methoxy,ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy,3-pentoxy, or hexyloxy; (C₂-C₆)alkenyl can be vinyl, allyl, 1-propenyl,2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl,3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or5-hexenyl; (C₂-C₆)alkynyl can be ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl,4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl;(C₁-C₆)alkanoyl can be acetyl, propanoyl or butanoyl; halo(C₁-C₆)alkylcan be iodomethyl, bromomethyl, chloromethyl, fluoromethyl,trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, orpentafluoroethyl; hydroxy(C₁-C₆)alkyl can be hydroxymethyl,1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl,3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl,5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl;(C₁-C₆)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, orhexyloxycarbonyl; (C₁-C₆)alkylthio can be methylthio, ethylthio,propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, orhexylthio; (C₂-C₆)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy,isobutanoyloxy, pentanoyloxy, or hexanoyloxy.

Therapeutic Methods

According to the invention, N-acetyl-D-mannosamine (ManNAc) andderivatives thereof are useful therapeutic agents for increasingproduction of sialic acids in mammals, and such increased production ofsialic acid has profound therapeutic benefits. Sialic acids areimportant for proper development and functioning of many organs andtissues, and a deficiency of sialic acid can give rise to many differenttypes of diseases and conditions. For example, abnormalities of sialicacid metabolism cause a severe infantile disease (infantile sialic acidstorage disease, ISSD), characterized by failure to thrive,hepatosplenomegaly, coarse facial features, severe mental and motorretardation presenting at birth and often leading to death within thefirst year of life, or diseases of later onset (Salla disease,sialuria).

As shown herein, administration of ManNAc (and derivatives thereof) isuseful for treating myopathies, muscular atrophy and/or musculardystrophy (e.g., hereditary inclusion body myopathy (HIBM)) and kidneyconditions and diseases (e.g., those involving proteinuria andhematuria).

Myopathies that can be treated with the present compositions and methodsalso include distal myopathy with rimmed vacuoles (Nonaka myopathy) andthe muscular dystrophy hereditary inclusion body myopathy (HIBM).

Proteinuria involves leakage of protein from the blood into the urine.If the amount of protein in the urine is very high, this condition isoften called nephrotic syndrome. While there may be many causes fornephritic syndrome, according to the invention at least one cause is adeficiency of sialic acid, which has a direct impact on the formation,structure and function of kidney glomeruli and the membranes associatedtherewith. Several types of diseases exhibit the symptoms ofproteinuria, including high blood pressure, infections, refluxnephropathy, diabetes, various types of glomerulonephritis, includingminimal change nephrosis. However, by improving the structure andfunction of nephron components that require sialic acid, the presentcompositions and methods can treat any of these diseases. Thus, forexample, the methods and compositions of the invention dramaticallyimprove kidney function by improving the structure and filtrationproperties of kidneys, thereby reducing the amount of protein in theurine and/or the severity or progression of proteinuria.

Hematuria simply means blood in the urine. The blood may be visible, sothat the urine appears reddish or darker than normal (called grosshematuria). If the blood is invisible and is discovered only when aurine sample is examined in a laboratory urine test, the condition iscalled microscopic hematuria. In general, hematuria is more a symptomthan a condition in itself, because it has many possible causes. Aurinary tract infection, kidney or bladder stones, an enlarged prostatein men, cystitis (a bladder infection, usually in women) or bladder,kidney or prostate cancer can all cause hematuria. Other causes includeinjuries that result in a bruised kidneys; sickle cell anemia and otherabnormal red blood cell diseases; and certain medications, such as bloodthinners (e.g., aspirin and some other pain relief medicines). Morespecific causes of glomerular basal membrane dysfunction, such as Alportdisease, thin membrane disease, and IgA nephropathy, may particularlyimprove when the treatment methods described herein are employed.

In general, the treatment methods of the invention involve administeringto a mammal (or patient) a therapeutically effective amount of N-acetylmannosamine and/or a derivative thereof. Such a therapeuticallyeffective amount is generally given daily for appropriate periods oftime. Effective amounts for human patients are, for example, about 0.1g/day to about 50 g/day, of about 0.2 g/day to about 25 g/day, fromabout 0.3 g/day to about 12 g/day, from about 0.4 g/day to about 10g/day, from about 0.5 g/day to about 8 g/day, and from about 0.7 g/dayto about 6 g/day. Generally, N-acetyl mannosamine and/or a derivativethereof is administered for periods of time sufficient to increase theamount of sialic acid in the mammal and thereby achieve a therapeuticbenefit. Therapeutic benefits that can be achieved by administration ofN-acetyl mannosamine and/or a derivative thereof include improved kidneyfunction, reduction in protein excretion in the urine, reduction inblood concentrations in the urine, increased sialylation of podocalyxin,increased sialylation of PSA-NCAM (and/or other tissue specific targetglycoproteins), fewer cystic tubular dilatations in the kidney cortexand in the kidney medulla, less fusion and flattening of the podocytefoot processes, greater number of open slit diaphragms in the kidneys,improvement in the “finger shaping” of the kidney foot processes,improved overall integrity of the GBM, increased Gne/Mnk proteinexpression and Gne-epimerase activities.

ManNAc is a ubiquitous but rare monosaccharide involved in a range ofmetabolic processes. It is uncharged and crosses membranes readily.ManNAc is a constituent of numerous glycolipids and glycoproteins, andis the first committed precursor for the biosynthesis ofN-acetylneuraminic (Neu5Ac, or sialic acid), which consists ofN-acetyl-D-mannosamine in an ether linkage with D-pyruvic acid. ManNAcis formed from UDP-N-acetylglucosamine (UDP-GlcNAc) by the action ofUDP-GlcNAc 2-epimerase. ManNAc is then phosphorylated by a specifickinase to ManNAc-6-P (FIG. 1). ManNAc is situated in the sialic acidbiosynthesis pathway after the regulated, rate-limiting GNE step (FIG.1), so its metabolism is not subject to feedback inhibition. ResidualMNK activity in HIBM patients, or ancillary kinases such as GlcNAckinase (Hinderlich et al. Eur. J. Biochem. 252: 133-139 (1998)), mightconvert ManNAc into ManNAc-6-phosphate for subsequent synthesis ofsialic acid. In fact, hyposialylated, Gne-deficient mouse embryonic stemcells became resialylated after their growth medium was supplementedwith ManNAc (Schwarzkopf et al. Proc. Natl. Acad. Sci. U.S.A. 99:5267-70 (2002)). Furthermore, incubation of cultured cells with“unnatural” ManNAc derivatives, i.e., N-levulinoylmannosamine (ManLev)or N-azidoacetyl-mannosamine (ManNAz), resulted in incorporation of thedownstream sialic acid analogs (SiaLev or SiaNAz) into cell surfaceglycoconjugates (Charter et al. Glycobiology 10: 1049-56 (2000)).

Hereditary Inclusion Body Myopathy (HIBM)

Studies of an Iranian-Jewish genetic isolate (Argov, et al., Neurology60, 1519-1523 (2003)) indicate that HIBM is mapped to chromosome9p12-13. The causative gene for HIBM is GNE, coding for the bifunctionalenzyme UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase.Eisenberg, et al. (2001) Nat Genet 29, 83-87 (2001); Tanner, M. E.,Bioorg Chem 33, 216-228 (2005); Stasche, et al. J Biol Chem 272,24319-24324 (1997); Hinderlich, et al. J Biol Chem 272, 24313-24318(1997); Jacobs, et al. Biochemistry 40, 12864-12874 (2001). The functionand feedback regulation of GNE/MNK is depicted in FIG. 1. DistalMyopathy with Rimmed Vacuoles (DMRV) is a Japanese variant, allelic toHIBM. Nishino et al. Neurology 59, 1689-1693 (2002); Kayashima et al. JHum Genet 47, 77-79 (2002); Hinderlich, et al. Neurology 61, 145 (2003).Nearly twenty GNE mutations have been reported in HIBM patients fromdifferent ethnic backgrounds, with founder effects among the IranianJews and Japanese. Broccolini et al. Hum Mutat 23, 632 (2004);Eisenberg, et al. Hum Mutat 21, 99 (2003); Tomimitsu, et al. Neurology59, 451-454 (2002); Darvish, et al. Mol Genet Metab 77, 252-256 (2002).The mutations causing HIBM occur in the regions encoding either theepimerase domain or the kinase domain. Most are missense mutations andresult in decreased enzyme GNE activity and underproduction of sialicacid. Sparks, et al. Glycobiology 15, 1102-1110 (2005); Penner, et al.Biochemistry 45, 2968-2977 (2006).

Sialic acids are negatively charged terminal sugar moieties added duringthe post-translational modification on oligosaccharide chains ofproteins and lipids to create glycoproteins and glycolipids. Varki,Faseb J 11, 248-255 (1997); Varki et al. Anal Biochem 137, 236-247(1984). They act as molecular determinants of specific biologicalprocesses such as cellular adhesion, cell-cell interactions and signaltransduction. Schauer, Glycoconj J 17, 485-499 (2000); Kelm et al. IntRev Cytol 175, 137-240 (1997).

The pathophysiology of HIBM remains largely unknown, but the dysfunctionin GNE suggests that impaired sialylation of glycoproteins is involved.Such a defect could influence cell-cell interactions, intracellulartrafficking, organelle biogenesis, apoptosis and secretion. In fact,UDP-GlcNAc 2-epimerase regulates sialylation of cell surface molecules(Keppler et al. Science 284: 1372-76 (1999)), and sialylation appears tobe critical for mouse development (Schwarzkopf et al. Proc Natl Acad SciUSA 99, 5267-5270 (2002)).

One hypothesis for the pathophysiology of HIBM involves undersialylationof α-DG, an essential component of the dystrophin-glycoprotein complex.Michele et al. Nature 418, 417-422 (2002); Michele et al. J Biol Chem278, 15457-15460 (2003). α-DG is heavily glycosylated with O-mannosylglycans (mannose-N-acetylglucosamine-galactose-sialic acid) linked to aserine or threonine; these glycans are critical for t-DG's interactionswith laminin and other extracellular ligands. Aberrant glycosylation ofα-DG is the underlying biochemical defect in several congenital musculardystrophies, generally termed “dystroglycanopathies,” includingFukuyama's congenital muscular dystrophy, Muscle-Eye-Brain disease,Walker-Warburg syndrome and the congenital muscular dystrophies type C1Cand C1D. Martin et al., Glycobiology 13, 67R-75R (2003); Martin-Rendon,et al. Trends Pharmacol Sci 24, 178-183 (2003). The inventors and othershave shown variable hyposialylation of α-DG and other glycoproteins,such as Neural Crest Adhesion Molecule (NCAM), in HIBM. Huizing et al.Mol Genet Metab 81, 196-202 (2004); Savelkoul et al. Mol Genet Metab 88,389-390 (2006); Sparks et al. BMC Neurol 7, 3 (2007); Broccolini et al.Neuromuscul Disord 15, 177-184 (2005); Ricci et al. Neurology 66,755-758 (2006); Salama et al. Biochem Biophys Res Commun 328, 221-226(2005); Tajima et al. Am J Pathol 166, 1121-1130 (2005).

However, prior to the present invention, the basic pathogenic mechanismsof HIBM, an HIBM animal model, and an effective therapy for HIBM werelacking. These issues are addressed by the present invention throughcreation of a Gne gene-targeted knockin mouse mimicking the M712Tmutation of Iranian-Jewish HIBM patients and through studies using thisknockin mouse model that have defined effective therapeutic methods.

A sequence for the mouse Gne protein is shown below (SEQ ID NO:1).

  1 MEKNGNNRKL RVCVATCNRA DYSKLAPIMF GIKTEPAFFE  41LDVVVLGSHL IDDYGNTYRM IEQDDFDINT RLHTIVRGED  81EAAMVESVGL ALVKLPDVLN RLKPDIMIVH GDRFDALALA 121TSAALMNIRI LHIEGGEVSG TIDDSIRHAI TKLAHYHVCC 161TRSAEQHLIS MCEDHDRILL AGCPSYDKLL SAKNKDYMSI 201IRMWLGDDVK CKDYIVALQH PVTTDIKHSI KMFELTLDAL 241ISFNKRTLVL FPNIDAGSKE MVRVMRKKGI EHHPNFRAVK 281HVPFDQFIQL VAHAGCMIGN SSCGVREVGA FGTPVINLGT 321RQIGRETGEN VLHVRDADTQ DKILQALHLQ FGKQYPCSKI 361YGDGNAVPRI LKFLKSIDLQ EPLQKKFCFP PVKENISQDI 401DHILETLSAL AVDLGGTNLR VAIVSMKGEI VKKYTQFNPK 441TYEERISLIL QMCVEAAAEA VKLNCRILGV GISTGGRVNP 481QEGVVLHSTK LIQEWNSVDL RTPLSDTLHL PVWVDNDGNC 521AAMAERKFGQ GKGQENFVTL ITGTGIGGGI IHQHELIHGS 561SFCAAELGHL VVSLDGPDCS CGSHGCIEAY ASGMALQREA 601KKLHDEDLLL VEGMSVPKDE AVGALHLIQA AKLGNVKAQS 641ILRTAGTALG LGVVNILHTM NPSLVILSGV LASHYIHIVK 681DVIRQQALSS VQDVDVVVSD LVDPALLGAA SMVLDYTTRR 721 IH

When this Gne protein has the M712T mutation, the sequence for Gnemutant protein arising from the Gne^(M712T) mutation is as follows (SEQID NO:2), where the methionine at position 712 has been changed to athreonine (bold and underlined amino acid shown below).

  1 MEKNGNNRKL RVCVATCNRA DYSKLAPIMF GIKTEPAFFE  41LDVVVLGSHL IDDYGNTYRM IEQDDFDINT RLHTIVRGED  81EAAMVESVGL ALVKLPDVLN RLKPDIMIVH GDRFDALALA 121TSAALMNIRI LHIEGGEVSG TIDDSIRHAI TKLAHYHVCC 161TRSAEQHLIS MCEDHDRILL AGCPSYDKLL SAKNKDYMSI 201IRMWLGDDVK CKDYIVALQH PVTTDIKHSI KMFELTLDAL 241ISFNKRTLVL FPNIDAGSKE MVRVMRKKGI EHHPNFRAVK 281HVPFDQFIQL VAHAGCMIGN SSCGVREVGA FGTPVINLGT 321RQIGRETGEN VLHVRDADTQ DKILQALHLQ FGKQYPCSKI 361YGDGNAVPRI LKFLKSIDLQ EPLQKKFCFP PVKENISQDI 401DHILETLSAL AVDLGGTNLR VAIVSMKGEI VKKYTQFNPK 441TYEERISLIL QMCVEAAAEA VKLNCRILGV GISTGGRVNP 481QEGVVLHSTK LIQEWNSVDL RTPLSDTLHL PVWVDNDGNC 521AAMAERKFGQ GKGQENFVTL ITGTGIGGGI IHQHELIHGS 561SFCAAELGHL VVSLDGPDCS CGSHGCIEAY ASGMALQREA 601KKLHDEDLLL VEGMSVPKDE AVGALHLIQA AKLGNVKAQS 641ILRTAGTALG LGVVNILHTM NPSLVILSGV LASHYIHIVK 681DVIRQQALSS VQDVDVVVSD LVDPALLGAA S T VLDYTTRR 721 IH

Although this M712T mutation gives rise to a recessive phenotype, it hasdramatic effects upon the survival and physiology of mammals. Forexample, HIBM exhibits non life-threatening symptoms in humans thatemerge in adulthood and lead to slowly progressive muscle weakness. Mostpatients develop symptoms while in their early 20s and becomewheelchair-bound by the time they reach 40, as their arm, hand, leg andcore muscles progressively weaken. The symptoms in mice are even moredramatic. For example, upon mating nine pairs of GneM^(712T/+) mice, 101progeny were obtained. Of those 101 progeny 26 homozygous mutated(Gne^(M712T/M712T)) mice were produced. However, only one male with theGne^(M712T/M712T) genotype survived past age P3 (FIG. 2D). The remaining25 Gne^(M712T/M712T) homozygous mutated offspring died at age P1-P3.This lone surviving mouse showed no muscle pathology at age P2. The lackof early myopathic features recapitulates the human HIBM phenotype. Inboth mice and humans, the muscle pathology occurs late or is attenuatedlikely by a modicum of sialic acid is provided through the actions ofresidual Gne/Mnk enzymatic activities (Sparks et al. Glycobiology 15:1102-10 (2005); Noguchi et al. J. Biol. Chem. 279: 11402-407 (2004))(FIG. 5F and FIGS. 6, A and B).

Kidney Conditions

Instead of early-onset muscle problems, homozygous Gne^(M712T/M712T)mice exhibit early signs of severe glomerular hematuria andpodocytopathy, including effacement of the podocyte foot processes andsegmental splitting of the glomerular basement membrane (GBM), likelydue to hyposialylation of specific membrane glycoproteins. Unexpectedly,the Gne^(M712T/M712T) knockin mice provide a novel animal model ofpodocytopathy and/or segmental splitting of the GBM, demonstrating thesignificance of sialic acid synthesis in kidney development andfunction. Structural elements in the kidney that are important forfiltering waste from the blood are severely impaired by the sialic aciddeficiency. This outcome demonstrates the significance of the ability ofthe body to synthesize sialic acid for kidney development and function.

As shown in the Examples and Figures of this application, administrationof ManNAc to pregnant mice had a remarkably salutary effect on thesurvival and renal development of homozygous pups. In particular, ManNAcadministration was associated with increased enzymatic activity of Gne,increased sialylation of kidney podocalyxin, and improved morphology ofthe podocyte foot processes and GBM integrity.

Therefore, according to the invention, ManNAc is effective not only as atreatment for HIBM but also for treatment of kidney disorders. Thus,ManNAc may be used to treat podocytopathies, minimal change nephrosis,focal and segmental glomerulosclerosis, membranous glomerulonephritis,and other forms of unexplained idiopathic nephrotic syndrome, as well asglomerular basement membrane diseases such as Alport disease and thinmembrane disease. Such kidney disorders and conditions are sometimescharacterized by segmental splitting of the glomerular basement membraneand/or podocytopathy due to disturbed polyanions on podocyte membranes,or to changes in the amount or charge (sialylation) of glomerularbasement membrane components.

Formulations and Administration

N-acetyl mannosamine and/or derivatives thereof are administered so asto achieve a reduction in at least one symptom associated with anindication or disease. For example, administration of N-acetylmannosamine and/or derivatives thereof can lead to a reduction inproteinuria (e.g., lower amounts of protein in the urine), a reductionin hematuria (e.g., lower amounts of red blood cells in the urine) andimprovement of muscle function (e.g., in patients with muscularatrophy).

To achieve the desired effect(s), N-acetyl mannosamine and/orderivatives thereof may be administered as single or divided dosages,for example, of at least about 0.01 mg/kg to about 500 to 750 mg/kg, ofat least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1mg/kg to about 200 to 400 mg/kg or at least about 1 mg/kg to about 25 to200 mg/kg of body weight, although other dosages may provide beneficialresults. The amount administered will vary depending on various factorsincluding, but not limited to the disease, the weight, the physicalcondition, the health, the age of the mammal, whether prevention ortreatment is to be achieved. Such factors can be readily determined bythe clinician employing animal models or other test systems that areavailable in the art.

Administration of the therapeutic agents in accordance with the presentinvention may be in a single dose, in multiple doses, in a continuous orintermittent manner, depending, for example, upon the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners. The administration of N-acetyl mannosamine and/orderivatives thereof may be essentially continuous over a pre-selectedperiod of time or may be in a series of spaced doses. Both local andsystemic administration is contemplated.

To prepare the composition, N-acetyl mannosamine and/or one or morederivatives thereof are synthesized or otherwise obtained, and purifiedas necessary or desired. N-acetyl mannosamine (and/or derivativesthereof) can then be added to a composition (or food product), adjustedto the appropriate concentration, and optionally combined with otheragents. The absolute weight of N-acetyl mannosamine and/or itsderivatives that is included in a unit dose can vary widely. Forexample, about 0.01 to about 2 g, or about 0.1 to about 1 g of N-acetylmannosamine and/or derivatives thereof are often used in compositions.Alternatively, the unit dosage can vary from about 0.01 g to about 50 g,from about 0.01 g to about 35 g, from about 0.1 g to about 25 g, fromabout 0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5g to about 4 g, or from about 0.5 g to about 2 g.

Daily doses of N-acetyl mannosamine and/or derivatives thereof can varyas well. Such daily doses can range, for example, from about 0.1 g/dayto about 50 g/day, from about 0.2 g/day to about 25 g/day, from about0.3 g/day to about 12 g/day, from about 0.4 g/day to about 10 g/day,from about 0.5 g/day to about 8 g/day, and from about 0.7 g/day to about6 g/day.

Thus, one or more suitable unit dosage forms comprising N-acetylmannosamine and/or derivatives thereof can be administered by a varietyof routes including oral, parenteral (including subcutaneous,intravenous, intramuscular and intraperitoneal), rectal, dermal,transdermal, intrathoracic, intrapulmonary and intranasal (respiratory)routes. The therapeutic agents may also be formulated for sustainedrelease (for example, using microencapsulation, see WO 94/07529, andU.S. Pat. No. 4,962,091). The formulations may, where appropriate, beconveniently presented in discrete unit dosage forms and may be preparedby any of the methods well known to the pharmaceutical arts. Suchmethods may include the step of mixing N-acetyl mannosamine and/orderivatives thereof with liquid carriers, solid matrices, semi-solidcarriers, finely divided solid carriers or combinations thereof, andthen, if necessary, introducing or shaping the product into the desireddelivery system.

When N-acetyl mannosamine and/or its derivatives is prepared for oraladministration, it is generally combined with a pharmaceuticallyacceptable carrier, diluent or excipient to form a pharmaceuticalformulation, or unit dosage form. For oral administration, N-acetylmannosamine (and/or derivatives thereof) may be present as a powder, agranular formulation, a solution, a suspension, an emulsion or in anatural or synthetic polymer or resin for ingestion of N-acetylmannosamine (and/or one or more derivatives thereof) from a chewing gum.The active ingredients may also be presented as a bolus, electuary orpaste. Orally administered N-acetyl mannosamine and/or derivativesthereof can also be formulated for sustained release. For example,N-acetyl mannosamine and/or derivatives thereof can be coated,microencapsulated, or otherwise placed within a sustained deliverydevice, for example, in order to avoid salivary bacteria degradation.The total N-acetyl mannosamine and its derivatives in such formulationscomprises from 0.1 to 99.9% by weight of the formulation.

By “pharmaceutically acceptable” it is meant a carrier, diluent,excipient, and/or salt that is compatible with the other ingredients ofthe formulation, and not deleterious to the recipient thereof.

Pharmaceutical formulations containing N-acetyl mannosamine and/orderivatives thereof can be prepared by procedures known in the art usingwell-known and readily available ingredients. For example, N-acetylmannosamine and/or its derivatives can be formulated with commonexcipients, diluents, or carriers, and formed into tablets, capsules,solutions, suspensions, powders, aerosols and the like. Examples ofexcipients, diluents, and carriers that are suitable for suchformulations include buffers, as well as fillers and extenders such asstarch, cellulose, sugars, mannitol, and silicic derivatives. Bindingagents can also be included such as carboxymethyl cellulose,hydroxymethylcellulose, hydroxypropyl methylcellulose and othercellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone.Moisturizing agents can be included such as glycerol, disintegratingagents such as calcium carbonate and sodium bicarbonate. Agents forretarding dissolution can also be included such as paraffin. Resorptionaccelerators such as quaternary ammonium compounds can also be included.Surface active agents such as cetyl alcohol and glycerol monostearatecan be included. Adsorptive carriers such as kaolin and bentonite can beadded. Lubricants such as talc, calcium and magnesium stearate, andsolid polyethyl glycols can also be included. Preservatives may also beadded. The compositions of the invention can also contain thickeningagents such as cellulose and/or cellulose derivatives. They may alsocontain gums such as xanthan, guar or carbo gum or gum arabic, oralternatively polyethylene glycols, bentones and montmorillonites, andthe like.

For example, tablets or caplets containing N-acetyl mannosamine (and/orits derivatives) can include buffering agents such as calcium carbonate,magnesium oxide and magnesium carbonate. Caplets and tablets can alsoinclude inactive ingredients such as cellulose, pre-gelatinized starch,silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate,microcrystalline cellulose, starch, talc, titanium dioxide, benzoicacid, citric acid, corn starch, mineral oil, polypropylene glycol,sodium phosphate, zinc stearate, and the like. Hard or soft gelatincapsules containing N-acetyl mannosamine (and/or its derivatives) cancontain inactive ingredients such as gelatin, microcrystallinecellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide,and the like, as well as liquid vehicles such as polyethylene glycols(PEGs) and vegetable oil. Moreover, enteric-coated caplets or tabletscontaining N-acetyl mannosamine and/or its derivatives are designed toresist disintegration in the stomach and dissolve in the more neutral toalkaline environment of the duodenum.

N-acetyl mannosamine and/or its derivatives can also be formulated as anelixir or solution for convenient oral administration or as a solutionappropriate for parenteral administration, for instance byintramuscular, subcutaneous, intraperitoneal or intravenous routes. Thepharmaceutical formulations of N-acetyl mannosamine and/or itsderivatives can also take the form of an aqueous or anhydrous solutionor dispersion, or alternatively the form of an emulsion or suspension orsalve.

Thus, N-acetyl mannosamine and/or its derivatives may be formulated forparenteral administration (e.g., by injection, for example, bolusinjection or continuous infusion) and may be presented in unit dose formin ampoules, pre-filled syringes, small volume infusion containers or inmulti-dose containers. As noted above, preservatives can be added tohelp maintain the shelve life of the dosage form. The N-acetylmannosamine, its derivatives and other ingredients may form suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the N-acetyl mannosamine, its derivatives andother ingredients may be in powder form, obtained by aseptic isolationof sterile solid or by lyophilization from solution, for constitutionwith a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

These formulations can contain pharmaceutically acceptable carriers,vehicles and adjuvants that are well known in the art. It is possible,for example, to prepare solutions using one or more organic solvent(s)that is/are acceptable from the physiological standpoint, chosen, inaddition to water, from solvents such as acetone, ethanol, isopropylalcohol, glycol ethers such as the products sold under the name“Dowanol,” polyglycols and polyethylene glycols, C₁-C₄ alkyl esters ofshort-chain acids, ethyl or isopropyl lactate, fatty acid triglyceridessuch as the products marketed under the name “Miglyol,” isopropylmyristate, animal, mineral and vegetable oils and polysiloxanes.

It is possible to add other ingredients such as antioxidants,surfactants, other preservatives, film-forming, keratolytic orcomedolytic agents, perfumes, flavorings and colorings. Antioxidantssuch as t-butylhydroquinone, butylated hydroxyanisole, butylatedhydroxytoluene and α-tocopherol and its derivatives can be added.

Additionally, N-acetyl mannosamine and/or derivatives thereof are wellsuited to formulation in a sustained release dosage form. Thus, suchformulations can be so constituted that they release the N-acetylmannosamine and/or its derivative, for example, in a particular part ofthe intestinal, urogenital or respiratory tract, over a period of time.Coatings, envelopes, and protective matrices may be made, for example,from polymeric substances, such as polylactide-glycolates, liposomes,microemulsions, microparticles, nanoparticles, or waxes. These coatings,envelopes, and protective matrices are useful to coat indwellingdevices, e.g., stents, catheters, peritoneal dialysis tubing, drainingdevices and the like.

For topical administration, N-acetyl mannosamine and/or itsderivative(s) may be formulated as is known in the art for directapplication to a target area. Forms chiefly conditioned for topicalapplication take the form, for example, of creams, milks, gels,dispersion or microemulsions, lotions thickened to a greater or lesserextent, impregnated pads, ointments or sticks, aerosol formulations(e.g., sprays or foams), soaps, detergents, lotions or cakes of soap.Other conventional forms for this purpose include wound dressings,coated bandages or other polymer coverings, ointments, creams, lotions,pastes, jellies, sprays, and aerosols. Thus, N-acetyl mannosamine and/orits derivatives can be delivered via patches or bandages for dermaladministration. Alternatively, N-acetyl mannosamine and/or itsderivatives can be formulated to be part of an adhesive polymer, such aspolyacrylate or acrylate/vinyl acetate copolymer. For long-termapplications it might be desirable to use microporous and/or breathablebacking laminates, so hydration or maceration of the skin can beminimized. The backing layer can be any appropriate thickness that willprovide the desired protective and support functions. A suitablethickness will generally be from about 10 to about 200 microns.

Ointments and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents, orcoloring agents. The therapeutic agents can also be delivered viaiontophoresis, e.g., as disclosed in U.S. Pat. No. 4,140,122; 4,383,529;or 4,051,842. The percent by weight of a therapeutic agent of theinvention present in a topical formulation will depend on variousfactors, but generally will be from 0.01% to 95% of the total weight ofthe formulation, and typically 0.1-85% by weight.

Drops, such as eye drops or nose drops, may be formulated with N-acetylmannosamine and/or derivatives thereof in an aqueous or non-aqueous basealso comprising one or more dispersing agents, solubilizing agents orsuspending agents. Liquid sprays are conveniently delivered frompressurized packs. Drops can be delivered via a simple eyedropper-capped bottle, or via a plastic bottle adapted to deliver liquidcontents dropwise, via a specially shaped closure.

N-acetyl mannosamine and/or its derivatives may further be formulatedfor topical administration in the mouth or throat. For example, N-acetylmannosamine and/or its derivatives may be formulated as a lozengefurther comprising a flavored base, usually sucrose and acacia ortragacanth; pastilles comprising the composition in an inert base suchas gelatin and glycerin or sucrose and acacia; and mouthwashescomprising the composition of the present invention in a suitable liquidcarrier.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that areavailable in the art.

Furthermore, N-acetyl mannosamine and/or its derivatives may also beused in combination with other therapeutic agents, for example, painrelievers, anti-inflammatory agents, and the like, whether for theconditions described or some other condition.

The present invention further pertains to a packaged pharmaceuticalcomposition such as a kit or other container for increasing productionof sialic acid in a mammal. The kit or container holds a therapeuticallyeffective amount of a pharmaceutical composition for increasingintracellular production of sialic acid and instructions for using thepharmaceutical composition for increasing production of sialic acid inthe mammal. The pharmaceutical composition includes N-acetyl mannosamineand/or its derivatives in a therapeutically effective amount such thatsialic acid production is increased.

Food Supplement

According to the invention, N-acetyl mannosamine and/or its derivativescan be administered as a food supplement or incorporated into food ordrink item such as a nutritional bar, snack bar, cookie, candy, cereal,pudding, ice cream, frozen confectionary, chewing gum, drink mix, sodapop, liquid supplement, sauce, salad dressing, gravy, jelly, jam,spread, margarine, peanut butter, nut spread, frosting, and the like. Inessence, can be used in any food, composition or supplement in whichsugar is employed. Hence, N-acetyl mannosamine and/or derivativesthereof can be used as a partial or full substitute for sugar.

Such food supplements, drinks and food items can include any other foodingredient including, for example, flour, oil, cream, butter, sugar,salt, spices and the like. In addition, the food supplements, drinks andfood items can include vitamins and nutrients commonly found in othernutritional supplements.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention.

Example 1: ManNAc Administration is Useful for Treating HIBM and RenalDisorders

This Example shows that ManNAc may be a useful treatment not only forHIBM but also for renal disorders involving proteinuria and hematuriadue to podocytopathy and/or segmental splitting of the glomerularbasement membrane.

Methods

Gne^(M712T/M712T) mice. Gne^(M712T/M712T) knockin mice were generated bytargeting the M712T (ATG to ACG) mutation of exon 12 of the murine Gnegene (Gne, Uae1, GenBank NM_015828) (FIG. 2A). The nucleotide sequencefor this Gne, Uae1 allele (GenBank NM_015828) without the mutation isshown below for easy reference (SEQ ID NO:3).

   1 GCTAAACCAG AGGCCAGACG GCAGCTCAGG AGTCCGACCA   41CACCTCAGGA AACAGCTGTG CCACAGGATG GAAACACACG   81CGCATCTCCA CAGGGAGCAG AGCTACGCAG GACCTCATGA  121ACTCTATTTT AAGAAACTCT CAAGTAAAAA GAAGCAAGTC  161ATGGAGAAGA ACGGGAACAA CCGAAAGCTC CGGGTTTGCG  201TTGCCACCTG CAACCGAGCT GACTACTCCA AACTGGCCCC  241GATCATGTTC GGCATCAAGA CAGAGCCCGC GTTCTTTGAG  281TTGGACGTGG TGGTGCTCGG CTCCCACCTG ATTGACGACT  321ATGGAAACAC ATACCGCATG ATTGAGCAAG ATGACTTTGA  361CATTAACACC AGGCTCCACA CGATTGTTAG AGGGGAAGAT  401GAAGCGGCCA TGGTAGAGTC GGTAGGCCTA GCGCTCGTGA  441AGCTACCGGA CGTCCTCAAT CGCCTGAAGC CCGACATCAT  481GATTGTTCAC GGAGACCGAT TTGACGCCCT TGCTCTGGCT  521ACGTCTGCTG CCTTGATGAA CATCCGCATC CTTCACATTG  561AAGGAGGCGA GGTCAGCGGG ACCATTGATG ACTCTATCAG  601ACACGCCATA ACAAAACTGG CTCACTACCA TGTGTGCTGC  641ACTAGAAGTG CAGAGCAGCA CCTGATCTCT ATGTGCGAGG  661ACCACGACCG CATCCTGTTG GCAGGCTGCC CTTCCTATGA  721CAAACTGCTC TCCGCCAAGA ACAAAGACTA TATGAGCATC  761ATTCGGATGT GGCTAGGCGA TGATGTAAAA TGTAAGGATT  801ACATCGTTGC CCTGCAGCAT CCCGTGACCA CTGACATTAA  841GCATTCCATA AAGATGTTTG AGCTAACACT GGATGCCCTG  881ATCTCGTTTA ACAAGAGGAC CCTAGTTCTG TTTCCAAATA  921TCGATGCAGG CAGCAAGGAG ATGGTTCGAG TGATGCGGAA  961GAAGGGCATC GAGCATCACC CCAATTTCCG TGCAGTCAAG 1001CACGTCCCGT TTGACCAGTT CATACAGCTG GTCGCCCACG 1041CTGGCTGCAT GATTGGGAAT AGCAGCTGCG GCGTGCGAGA 1081GGTTGGCGCT TTCGGAACAC CCGTGATCAA CCTGGGCACA 1121AGGCAGATAG GAAGAGAAAC CGGGGAGAAT GTTCTTCATG 1161TCAGGGATGC TGACACCCAA GATAAAATAT TGCAAGCACT 1201ACACCTCCAG TTCGGCAAAC AGTACCCTTG CTCAAAGATA 1241TATGGGGATG GGAATGCTGT TCCAAGGATT TTAAAGTTTC 1281TCAAATCCAT TGACCTTCAA GAGCCACTAC AGAAGAAATT 1321CTGCTTCCCC CCTGTAAAGG AGAACATCTC TCAAGACATT 1361GACCACATCC TGGAAACTCT GAGTGCCTTG GCTGTTGATC 1401TTGGCGGGAC AAACCTGAGG GTGGCAATAG TTAGCATGAA 1441GGGTGAAATC GTTAAGAAGT ACACTCAGTT CAACCCTAAA 1481ACCTATGAAG AAAGGATTAG TTTAATCCTG CAGATGTGTG 1521TGGAAGCTGC CGCGGAAGCT GTGAAACTCA ATTGCAGAAT 1561TCTGGGAGTA GGCATCTCCA CAGGTGGCCG CGTGAATCCC 1601CAGGAAGGAG TTGTGCTGCA TTCAACCAAG CTGATCCAGG 1641AATGGAACTC CGTGGACCTC AGGACACCCC TCTCCGACAC 1681CCTGCATCTC CCCGTGTGGG TGGACAATGA CGGCAACTGT 1721GCCGCCATGG CAGAGAGGAA GTTCGGCCAA GGAAAAGGAC 1761AGGAGAACTT CGTGACGCTC ATCACGGGGA CAGGGATCGG 1801TGGGGGGATC ATCCACCAGC ACGAACTGAT CCACGGCAGC 1841TCCTTCTGCG CGGCGGAGCT CGGCCATCTC GTGGTGTCCC 1881TGGACGGTCC TGACTGCTCC TGTGGAAGCC ATGGGTGCAT 1921CGAAGCGTAC GCCTCTGGAA TGGCCTTGCA GAGGGAAGCA 1961AAGAAACTCC ATGATGAGGA CCTGCTCTTG GTGGAAGGGA 2001TGTCAGTACC AAAAGACGAA GCTGTGGGTG CCCTCCATCT 2041CATCCAGGCT GCCAAGCTGG GCAACGTGAA GGCCCAGAGC 2081ATCTTACGAA CAGCTGGAAC TGCTTTGGGA CTTGGGGTTG 2121TGAACATCCT CCACACTATG AATCCTTCCC TGGTGATCCT 2161GTCTGGAGTC CTGGCCAGTC ACTACATCCA CATCGTGAAG 2201GACGTCATCC GCCAGCAAGC CTTGTCCTCC GTGCAGGATG 2241TGGACGTGGT GGTCTCAGAC TTGGTGGACC CGGCCCTGCT 2281TGGCGCAGCC AGCATGGTTC TGGACTACAC AACGCGCAGG 2321ATCCACTAGG TCTCCCGGGA ACGGACACGG ACAGAGACTC 2361GGGAGCTGCT TAGAGTGGAA CCATGCTCTT CTAGATCAGT 2401GTTTCTGCGA AGGCAAATTT GGGGGGAGGG CTGCTGAGAC 2441AGCTCAGTGG TTAAGAGCCT GCCCTGCTCC TGCCAGTCCC 2481CAGCACCCAT GTCAGGCAGC TCAGCTGCCT GGAAGCCAAG 2521CTCCAGGGGA CCCAATGCCT CTCTGCCGGG GGCAGCTGCA 2561CTCAGATGTA CATACCCCTC TCCACACACA TACAAATAAA 2601GCTTATTTTT CAAAAGGCAA AAAAAAAAAA AAAAAAAAAA 2641 AAAAAAAAAA AAAAThe mutant mice were maintained in the C57BL/6J background. Animals werehoused in an accredited specific pathogen-free facility in accordancewith accepted guidelines. Cages were ventilated in a temperature- andlight-controlled environment (22° C., 30%-70% humidity, 12-hourlight/12-hour dark cycle). The mice were fed irradiated chow (Prolab5P75 Isopro 3000; PMI Nutrition International) and sterile water adlibitum. All euthanasia was performed by CO₂ inhalation followed bycervical dislocation.

For Mendelian distribution studies, 4 pregnant mice at E17-E19 wereeuthanized, and embryos were retrieved by cesarean section andeuthanized by decapitation. All mouse procedures were performed inaccordance with protocol G04-3 and were approved by the InstitutionalAnimal Care and Use Committee of the National Human Genome ResearchInstitute.

Molecular Analysis.

Mouse genotyping was performed on tail genomic DNA or cDNA isolated fromkidney or skeletal muscle using standard protocols. Total RNA wasisolated from murine tissues using the TRIzol reagent (Invitrogen), andcDNA was prepared using the SuperScript III system (Invitrogen). PCRamplifications were performed across the M712T mutation with genomic DNAas template, using the primer set 5′-agcacttcctgagtttgatg-3′ (SEQ IDNO:4) and 5′-atttgccttcgcaga-cacttga-3′ (SEQ ID NO:5) (FIG. 2B) or withcDNA as template (FIG. 2C), using the primer set5′-GCCCAGAGCATCTTACGAAC-3′ (SEQ ID NO:6) and 5′-GGGTCCCCTGGAGCTTGG-3′(SEQ ID NO:7) and PuReTaq Ready-To-Go PCR beads (GE Healthcare), usingstandard PCR conditions. PCR fragments were digested with Nla III at 37°C. to verify the mutation status (FIGS. 2B and C). Quantitative realtimePCR was performed on RNA isolated from kidney and skeletal muscle,utilizing Assays-On-Demand (Applied Biosystems) for Gne (mm00607939),Pecam-1 (mm00476702), Col4A3 (mm01269206), and β-actin (mm00450174) onan ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems).

Clinical Chemistry Screen.

Retroorbital blood samples (100-150 ml) from weaned mice (weighing atleast 15 grams) matched for sex (male) and age were obtained bimonthlyafter pretreatment with a topical anesthetic (0.5% tetracaine HCl;Bausch & Lomb Pharmaceuticals). Samples were allowed to clot (30 minutesat room temperature) in MicroPrep centrifuge tubes (IRIS InternationalInc.), after which the serum was separated by centrifugation at 1500 gfor 10 minutes and stored at −80° C. until analysis. Clinical chemistryscreens were performed at the Department of Laboratory Medicine at theNIH and included monitoring of creatinine, blood urea nitrogen, albumin,total protein, uric acid, alkaline phosphatase, alanineaminotransferase, aspartate aminotransferase, amylase, creatine kinase,and lactate dehydrogenase. In addition, reagent strips for proteinurinalysis were used to assess proteinuria (Chemstrip 2GP; RocheDiagnostics).

Antibodies.

A rabbit polyclonal antibody was custom prepared against a Gne/Mnkpeptide comprising amino acids 588-607 (EAYASGMALQREAKKLHDED, SEQ IDNO:8), coupled to keyhole limpet hemocyanine, and affinity purifiedagainst the corresponding antigenic peptide (Covance). The followingadditional primary antibodies were commercially obtained: dystrophin(catalog no. ab15277; Abcam); α-dystroglycan (clone IIH6C4; UpstateBiotechnology); laminin-1 (catalog no. L9393; Sigma-Aldrich);podocalyxin (catalog no. PODX11-A; Alpha Diagnostic International);podocin (catalog no. P0372; Sigma-Aldrich); laminin P31 (catalog no.MAB1928; Millipore); desmin (catalog no. 1466-1; Epitomics); α-SMA(SPM332; GeneTex Inc.); PSA-NCAM (catalog no. MAB5324; Millipore); andβ-actin (catalog no. AAN01; Cytoskeleton).

Mouse Histology.

Mouse tissues were collected, formalin fixed (10%) and paraffinembedded. Tissue sections (5 mm) were stained with H&E followingstandard procedures (American Histolabs) or subjected toimmunohistochemistry with a variety of primary antibodies.Formalin-fixed tissues were deparaffinized in HistoClear II (NationalDiagnostics) and dehydrated in a series of ethanol solutions. Antigenretrieval was performed for sections that were to be stained withantibodies against Gne/Mnk (by boiling 5 minutes in citric acid-basedsolution; Vector Laboratories) and against dystrophin (by boiling in 1mM EDTA according the manufacturer's protocol; AbCam). The sections wereblocked (2% BSA, 10% donkey serum, and 0.1% Triton X-100 in PBS) andincubated with primary antibodies (Gne/Mnk 1:50; laminin 1:25;dystrophin 1:50) overnight at 4° C., followed by incubation with thesecondary antibody, Alexa Fluor 488-conjugated donkey anti-rabbit (1:500in blocking solution) (Invitrogen). The sections were mounted inVECTASHIELD Mounting Medium (Vector Laboratories) and viewed anddigitally imaged with a Zeiss Axiovert 200M microscope (Zeiss).

Western Blotting.

Mouse tissues (age P2) were extracted, homogenized in CelLytic bufferconsisting of a mild detergent, bicine buffer, and 150 mM NaCl(Sigma-Aldrich) supplemented with protease inhibitors (Complete Mini;Roche Applied Science). The lysates were sonicated and cleared bycentrifugation (1000 g for 10 minutes), and the resulting supernatantswere assayed for protein (BCA Protein Assay; Pierce Biotechnology). Forthe neuraminidase enzymatic treatments (FIG. 6E), protein homogenates(25 mg) were incubated for 30 min at 37° C. with 1 mU/mg ofneuraminidase (catalog no. N6514; Sigma-Aldrich). Equal amounts ofprotein (25-50 mg) were electrophoresed on 4%-12% Tris-Glycine gels(Novex; Invitrogen), and electroblotted onto 0.45 mm Hybond ECLnitrocellulose membranes (GE Healthcare). The membranes were blocked(10% fat-free milk) and incubated with primary antibodies followed byHRP-conjugated secondary antibodies (GE Healthcare). Results werevisualized with ECL (ECL Western Blotting Detection Reagents; GEHealthcare) and exposure to CL-XPosure Film (Pierce Biotechnology).Densitometry was performed on the digital images obtained with a KodakImage Station and software (PerkinElmer). The protein levels werenormalized to those of β-actin to correct for differences in proteinloading and/or transfer.

Electron Microscopy.

Kidney samples were fixed overnight at 4° C. in 2% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4) and washed with cacodylate buffer. Afterpostfixation with 1% OsO₄ for 2 hours and a second wash with 0.1 Mcacodylate buffer, the tissues were serially dehydrated in ethanol andembedded in Eponate 12 resin (Ted Pella). Thin sections (˜80 nm) wereobtained using a Leica Ultracut UCT Ultramicrotome (Leica Microsystems),placed onto 400 mesh copper grids, and stained with saturated uranylacetate in 50% methanol followed by lead citrate. The grids were viewedwith a Philips 410 electron microscope (FEI Company) at 80 kV, andimages were recorded on Kodak SO-163 film (Kodak).

ManNAc Administration.

Breeding pairs of 6-week-old GneM^(712T/+) mice were divided into 3groups. Group I consisted of 9 GneM^(712T/+) breeding pairs, who wereadministered untreated sterilized tap water. Group II consisted of 1breeding pair of Gne^(+/+) mice (wild-type control) and 6 GneM^(712T/+)breeding pairs, who were administered water containing 1 mg/ml (˜0.2g/kg/day) ManNAc (Sigma-Aldrich). That dose was selected based onprevious evidence of the safety of ManNAc (administered at a single doseof 0.142 g/kg/day) in a study performed in humans (21). Group IIIconsisted of 1 Gne^(+/+) breeding pair and 7 GneM^(712T/+) breedingpairs, who were administered water supplemented with 5 mg/ml (˜1.0g/kg/day) ManNAc. Water was changed twice weekly. Nursing femalescontinued to be supplied with ManNAc. All mice were weaned from ManNAcat 21 days. Selected whole litters were euthanized at age P2, P6, andP19 for histological, genetic, biochemical, or ultrastructural analysis.

Gne Enzymatic Assays.

Mouse kidney and skeletal muscle (quadriceps) tissues were homogenizedand subjected to the Gne-epimerase enzymatic assay as previouslydescribed (11, 58). This assay was based on incubation with radiolabeledsubstrate (Uridine diphosphate N-acetyl glucosamine [1-³H]; AmericanRadiolabeled Chemicals Inc.) and detection of radiolabeled product([³H]ManNAc) upon separation of oligosaccharides by high-pHanion-exchange chromatography with pulsed amperometric detection(Dionex).

Statistics.

Differences between data groups were evaluated for significance usingthe 2-tailed Student's t test of unpaired data. For Mendeliandistribution analysis, a goodness-of-fit (χ2) test was performed, whilefor comparisons of survival between treated and untreated mice of allgenotypes (Gne^(+/+), GneM^(712T/+), and GneM^(712T/M712T)), a 2-tailedFisher's exact test using a 2×3 table was employed. All data arepresented as the mean±SD. A P value less than 0.05 was consideredstatistically significant.

Results

Generation of Gne^(M712T/M712T) Knockin Mice.

A murine targeting vector for homologous recombination in C57BL/6Jembryonic stem cells was constructed to include the M712T Gne mutation(FIG. 2A). The neomycin phosphotransferase and thymidine kinase geneswere introduced into the vector as positive and negative selectionmarkers, respectively (FIG. 2A). Additional LoxP (flanking exon 12 andneo) and flippase recombinase target sites (flanking neo) were insertedfor transgenic models for this condition (Nagy, A. 2000. Crerecombinase: the universal reagent for genome tailoring. Genesis.26:99-109). The entire vector was sequence verified. Genotyping of themice was performed by PCR amplification and digestion with therestriction endonuclease NlaIII (FIG. 2B). Tissues of homozygous mutantGne^(M712T/M712T) and wild-type Gne^(+/+) mice showed comparable Gne RNAtranscript levels by real-time quantitative PCR. Furthermore, NlaIIIdigestion of amplified cDNA demonstrated homozygous insertion of theM712T mutation in RNA of Gne^(M712T/M712T) mice (FIG. 2C).

Early Postnatal Lethality.

Initial matings of heterozygous mice (Gne^(M712T/+)) yielded 101offspring from which only 1 Gne^(M712T/M712T) animal survived beyondP21. The remaining Gne^(M712T/M712T) offspring died at P1-P3 (FIG. 2D).However, subsequent genotyping of 35 embryos at days E17-E19 showed 26%Gne^(+/+), 43% GneM^(712T/+), and 31% GneM^(712T/M712T), reflecting aMendelian distribution, statistically confirmed by goodness-of-fittesting (12=0.94, P=0.62) (FIG. 2D). At E17-E19, the embryos displayednormal exteriors, normal head and body sizes, and pink skin, whichindicated good circulatory and respiratory function. By P2, however,Gne^(M712T/M712T) mice were smaller than control littermates (FIG. 2E),weighing 70%-100% of control littermates. The Gne^(M712T/M712T) mousestomachs contained milk, although a prominent milkspot was not alwaysvisible. All Gne^(M712T/M712T) mice except 1 died by P3 and hadincreased urinary protein. In contrast, Gne^(M712T/+) mice appearedunaffected.

Histological Analyses.

Tissues of Gne^(M712T/M712T) mice and their littermates were examinedbetween age P2 and P3. No abnormalities were identified in skeletalmuscle, heart, or liver (data not shown). Moreover, immunohistochemicalstaining with antibodies against laminin and dystrophin failed to showdifferences between muscle sections of Gne^(M712T/M712T) mice and theirwild-type littermates.

At age P2, kidneys of Gne^(M712T/M712T) mice showed petechialhemorrhages by gross examination, but were normal in size and shapecompared with kidneys of Gne^(+/+) and Gne^(M712T/+) littermates (FIG.3A). Histological analyses revealed cystic tubular dilatation (FIG. 3B).High-magnification views of Gne^(M712T/M712T) kidneys showed red bloodcell infiltrates in the proximal and distal convoluted tubules and thecollecting ducts (FIG. 3C). The glomeruli of Gne^(M712T/M712T) micecontained red blood cell infiltrates in Bowman space (FIG. 3D). Of 100glomeruli scored in each group, 64%±6% were affected inGne^(M712T/M712T) mice (n=4) compared with 2%±1% in Gne^(M712T/+) mice(n=3) and 4%±4.5% in Gne^(+/+) mice (n=4). Immunohistochemical analysisdemonstrated localization of Gne/Mnk antibodies to kidney glomeruli(FIG. 3E). Examination of Gne^(M712T/M712T) kidneys at E18 showed nohistological differences compared with wild-type or heterozygouslittermates (data not shown).

Ultrastructural analyses of the glomeruli at age P2 revealed that,compared with the slender, well-shaped glomerular foot processes ofwild-type mice (FIG. 4A), the podocyte foot process membranes ofGne^(M712T/M712T) mice were flattened and largely fused, with only a fewwide foot processes remaining (FIG. 4B). Filtration slits were reducedin number and showed formation of tight junction-like structures (FIG.4B). In addition, the GBM showed segmental splitting of the lamina densa(FIG. 4B). The size and shape of endothelial cells lining the basementmembrane, as well as glomerular mesangial cells, appearedultrastructurally intact.

To support these ultrastructural findings, additional analyses wereperformed using markers for specific glomerular compartments. Thepodocyte-specific markers podocin and podocalyxin (Pavestadt et al.,Physiol. Rev. 83: 253-307 (2003); Dekan et al. Proc. Natl. Acad. Sci.USA 88: 5398-5402 (1991)) were tested by immunoblotting kidney extractsof all genotypes. While podocin showed no difference in expressionacross all genotypes (at age P1) (data not shown), podocalyxin, themajor sialoglycoprotein of the podocyte apical membrane (Pavestadt etal.; Dekan et al.), demonstrated dramatically decreased sialylation(FIG. 6E, upper gel). Expression levels of GBM markers laminin-1 (FIG.6C) and laminin 131 (data not shown) were unchanged in Gne^(M712T/M712T)kidneys, as were RNA levels of collagen type IV α3 (Col4A3), an integralGBM component. Immunoblotting with desmin and vascular SMA, antibodiesto mesangial cell markers (Ichimura et al. J. Histochem. Cytochem. 54:1291-1301 (2006)), showed similar expression levels across allgenotypes. In addition, real-time quantitative PCR analysis of theendothelial cell marker CD31/Pecam-1 revealed no difference in RNAexpression levels across genotypes at P1. Serum metabolite studies onthe only Gne^(M712T/M712T) mouse that survived past weaning demonstratedelevated blood urea nitrogen levels (39±10 mg/dl in Gne^(M712T/M712T)mouse versus 21±2 mg/dl in Gne^(+/+) mice) and increased urinary protein(>500 mg/dl protein), which indicated renal disease. All other serummetabolites tested, including creatinine and creatine kinase, werewithin the normal ranges. This male GneM^(712T/M712T) survivor waseuthanized at age 8.5 months. Histologic analysis revealed no structuralabnormalities in the forelimb or hindlimb. However, severe bilateralhydronephrosis and changes consistent with glomerulopathy were found inthe kidneys.

Rescue by ManNAc Feeding.

ManNAc, added to the drinking water at a concentration of 1 mg/ml (˜0.2g/kg/day) during matings of GneM^(712T/+) mice, yielded no survivinghomozygous GneM^(712T/M712T) mice beyond age P3 from among 51 offspring(FIG. 5A). However, at 5 mg ManNAc/ml (˜1.0 g/kg/day), among 102 totalnewborns, 12 Gne^(M712T/M712T) pups survived beyond P3, a significantlygreater number compared with the 1 survivor in the untreated group(2-tailed Fisher's exact test, P=0.01) (FIG. 5A). ManNAc at theadministered dose (˜1.0 g/kg/d) was well tolerated by the mice, and noside effects were attributed to the treatment throughout the study.Surviving Gne^(M712T/M712T) mice remained smaller than their wild-typelittermates, weighing 70%-100%. At age P6, ManNAc treatedGne^(M712T/M712T) mice exhibited no abnormalities in liver, heart, orskeletal muscle tissues (data not shown). Their kidneys demonstratedsignificant histological improvement (FIG. 5B-D) compared withGne^(M712T/M712T) mice examined at age P2 (FIG. 3B-D). Upon ManNActreatment, there were fewer cystic tubular dilatations in the cortex andmedulla (FIG. 5B) and reduced red blood cell infiltrates in the tubulesand the Bowman space (FIGS. 5C and D). Ultrastructural analysis at ageP19 showed less fusion and flattening of the podocyte foot processesincluding a greater number of open slit diaphragms and an improvement inthe “finger shaping” of the foot processes (FIGS. 4C and D). The overallintegrity of the GBM was also significantly improved, althoughoccasional segmental splitting of the lamina densa was still apparent(FIGS. 4C and D).

The nursing females continued to receive ManNAc treatment until the pupswere weaned (P21). Of the twelve Gne^(M712T/M712T) mice that survivedpast P3, nine died between P6 and P12. One Gne^(M712T/M712T) mouse wassacrificed at age P19 for ultrastructural analysis. TwoGne^(M712T/M712T) mice survived past P21, when ManNAc supplementationwas ceased. These two mice continued to grow without receivingadditional ManNAc but remained smaller than their littermates (FIG. 5E).At 3.5 months of age, one Gne^(M712T/M712T) survivor was sacrificedbecause of hydrocephalus and malocclusion. Similar events occurred insome untreated mice at different ages and were found not to be relatedto treatment or the disease. Skeletal muscle histology of this mouserevealed no structural or inflammatory abnormalities, but the kidneysshowed mild red blood cell infiltrations in the urinary space and thetubules. The one surviving Gne^(M712T/M712T) mouse is currently 6 monthsold and has no obvious myopathic features.

Biochemical Analyses Following ManNAc Feeding.

Gne enzymatic activity was measured in muscle and kidney at age P2.Skeletal muscle of Gne^(M712T/M712T) mice showed 19.4%±7.5 of the Gneactivity of the Gne+/+ mice (n=4, P=0.02) (FIG. 5F). Similar decreasesin Gne activities were measured in Gne^(M712T/M712T) kidney extracts(10% of mean Gne^(+/+) kidney epimerase activities). Upon ManNActreatment, Gne activities in Gne^(+/+) muscle (n=3) increased to 114%(±19.7) (P=0.2), while Gne^(M712T/M712T) muscle activity (n=7) increasedfrom 19.4% (±7.5) to 31% (±8.4) of untreated Gne^(+/+) mean values ofmuscle Gne activity (P=0.05) (FIG. 5F). Immunoblots of muscle and kidneyextracts labeled with anti-Gne/Mnk antibodies demonstrated 38.5% (±27,n=4) Gne/Mnk protein in Gne^(M712T/M712T) muscle and 32.1% (±7, n=3) inGne^(M712T/M712T) kidney tissues when compared with Gne^(+/+)littermates. This improved upon ManNAc treatment of Gne^(M712T/M712T)mice to 68.8% (±20, n=4) in muscle and to 62.2% (±9.7, n=4) in kidneytissues (P=0.12 and P=0.006 for muscle and kidney values respectively,relative to β-actin) (FIGS. 6A and B). Immunoblots stained withantibodies against laminin-1, an integral component of the GBM (25-27),showed similar patterns across genotypes before and after treatment(FIG. 6C).

The degree of sialylation of two heavily sialylated marker proteins,PSA-NCAM and podocalyxin was evaluated. PSA-NCAM is a major sialoproteinexpressed in neonatal brains (Galuska et al., J. Biol. Chem. 281:31605-15 (2006)), where its expression is regulated by the intracellularconcentration of sialic acid (Bork et al., FEBS letters 579: 5079-83(2005)).

FIG. 6 shows that the expression of PSA-NCAM varied within and betweengenotypes, yet Gne^(M712T/M712T) brains at P2 showed up to 80% decreasedPSA-NCAM expression compared with that in Gne+/+ mice (FIG. 6D, uppergel). A 2%-28% increase compared with Gne^(M712T/M712T) untreated micefollowing ManNAc treatment was observed (n=14 before treatment and n=10after treatment, P=0.08) (FIG. 6D, lower gel). The expression ofPSA-NCAM in normal muscle and kidney at P2 was low, and no change upontreatment in these tissues could be confirmed (data not shown). Inaddition, the significantly decreased sialylation status of podocalyxinin untreated Gne^(M712T/M712T) kidneys (FIG. 6E, lower gel) markedlyimproved upon ManNAc treatment (FIG. 6E, upper gel).

Therefore, this Example describes a knockin mice harboring a M712TGne/Mnk mutation that was generated by the inventors. Homozygous mutant(GneM^(712T/M712T)) mice did not survive beyond P3 unless treated withManNAc. At P2, significantly decreased Gne-epimerase activity wasobserved in Gne^(M712T/M712T) muscle, but no myopathic features wereapparent. Rather, homozygous mutant mice had glomerular hematuria,proteinuria, and podocytopathy. Renal findings included segmentalsplitting of the glomerular basement membrane, effacement of podocytefoot processes, and reduced sialylation of the major podocytesialoprotein, podocalyxin. ManNAc administration led to survival beyondP3 in 43% of the Gne^(M712T/M712)T pups. Survivors exhibited improvedrenal histology, increased sialylation of podocalyxin, and increasedGne/Mnk protein expression and Gne-epimerase activities. These findingsindicate that ManNAc may be a useful treatment not only for HIBM butalso for renal disorders involving proteinuria and hematuria due topodocytopathy and/or segmental splitting of the glomerular basementmembrane.

Example 2: Administration to Humans—Clinical Studies

Patients will be recruited from the Iranian Jewish community, fromgroups of patients known to have HIBM, and from individuals previouslyenrolled in protocol 76-HG-0238, “Diagnosis and Treatment of Patientswith Inborn Errors of Metabolism,” protocol 01-N-0149, “DiagnosticEvaluation of Patients with Neuromuscular Diseases,” or protocol05-HG-0236, “Pilot Study of the Use of Intravenous Immune Globulin inHereditary Inclusion Body Myopathy.” Patients will also be recruitedfrom the patient organization ARMS (Advancement of Research forMyopathies).

Materials

ManNAc for human use will be purchased from Meropharm AG(Eugensbergstrasse 14, 8268 Salenstein, Switzerland). It will beprepared as 500 mg enteric coated capsules by the Clinical CenterPharmaceutical Development Service (PDS), who will also prepare aplacebo containing mannose. ManNAc or placebo will be taken orally infour divided doses, thirty minutes before meals. ManNAc and placebocapsules will be delivered monthly for each study patient and stored inthe refrigerator. All bottles will be labeled in the same way and therewill be no apparent difference between ManNAc and placebo capsules incolor, shape or taste of the coating.

Procedures

Baseline evaluations will include a detailed determination of the extentof neuromuscular disease prior to treatment. The history and physicalexamination will include elucidation of the family pedigree,neurological status, and muscle strength. Baseline laboratory tests willalso include a routine urinalysis. Women will receive a pregnancy test.Blood will be drawn for CBC and differential, platelets, erythrocytesedimentation rate, electrolytes, calcium, phosphorus, liver enzymes,lipid panel, alkaline phosphatase, prothrombin time, partialthromboplastin time, creatine phosphokinase, HbA1C, fasting glucose andinsulin, free T4 and TSH, FSH, LH, testosterone and estradiol. A purpletop tube will be obtained for DNA/RNA extraction, a yellow top forlymphoblast transformation and separate yellow and brown tops forplatelet and white cell pellets. The DNA/RNA will be used to perform orconfirm mutational analysis of the GNE gene. The cells will be used forbasic research studies, including assessment of the sialylation statusof glycoproteins. Blood and urine will also be obtained fordetermination of serum and urine ManNAc and free sialic acid levels. Inaddition to spot urine tests, a 24-hour urine collection will beanalyzed for creatinine clearance, protein, albumin, proteinelectrophoresis, 12-microglobulin, and amino acid analysis. Baselineblood tests and their volumes are as follows:

Total Volume # Drawn ml/Test (ml) CBC, ESR, platelets 1 3.0 3.0 Chem 20panel, CPK, 1 3.5 3.5 & fasting glucose and lipid panel PT, PTT 1 4.54.5 HbA1C, Insulin 1 3.5 3.5 Free T4/TSH 1 3.5 3.5 FSH, LH, Test,,Estradiol 1 5.0 5.0 Lymphoblasts 1 5.0 5.0 Platelet pellet 1 5.0 5.0Leucocyte pellet/amino acids 1 5.0 5.0 DNA/RNA 1 8.0 8.0 Serum ManNAc,SA 2 4.0 8.0 Total 54.0In addition, up to 30 ml of blood may be removed for research purposes.However, under no circumstances will more than 450 ml of blood bewithdrawn during any 6-week period. Patients' saliva will also becollected. In addition, a radiograph of the chest, an echocardiogram andan electrocardiogram and a 24h ambulatory ECG will be obtained.

The primary outcome parameter will be a change in quadriceps musclestrength, as well as secondary outcome parameters. Maximal voluntaryisometric contraction (MVIC) assessment will be used to measure changesin the strength of the quadriceps muscles and 10 other muscle groups.MVIC assessment has proven reliable and sensitive to small changes inthe evaluation of myopathy syndromes and their response to treatment.For MVIC, the quantitative muscle assessment (QMA) system-version 42+XLwill be used. This system consists of an adjustable strap, attaching thelimb to an interface SM250 force transducer. Patients will be tested onan adjustable examining table (Neurological Plinth Model), enclosed in astable aluminum frame that anchors the transducer. The generated forceis transmitted to an electronic strain-gauge tensiometer and is thenrecorded and amplified by the computer-assisted analog/digital datacollection system (S/N A98C36). The measured force is expressed as theamount of kilograms (kg) that the patient exerts against the straingauge.

Measurements will be performed by standardized testing modified from theprotocol of Andres et al. (Neurology 36, 937-941 (1986)). The followingmuscle groups will be tested in a fixed order: right shoulder abductors,right elbow flexors, right elbow extensors, left shoulder abductors,left elbow flexors, left elbow extensors, right ankle dorsiflexors, leftankle dorsiflexors, right knee flexors and left knee flexors, left kneeextensors, right knee extensors. All muscle groups will be tested twicewith a minimum of 30 seconds rest between the trials. If measurementsdiffer by more than 15% a third test will be performed. The average ofthe two more similar measurements will be used as the score for thatparticular test. The patients will be informed about the test purposeand procedure before the start of the study and verbal encouragementwill be given during the tests.

Patients will also perform a 6-minute walk test, a timed up-and-go test,measures of pinch and grip strength, and the forward/functional reachtest.

Skeletal muscle strength will also be measured by physical examination.The 10-point manual muscle testing (MMT-28) scale (Jain et al. PhysOccup Ther Pediatr 26, 5-17 (2006)), in which 0 is the lowest and 10 thehighest score, will be used for grading of the response. The MMT will beperformed by a physical therapist who will remain blinded and willinclude physical examination of the following 13 muscle groups on eachside: deltoid, biceps brachii, triceps brachii, brachioradialis, wristextensors, wrist flexors, iliopsoas, gluteus maximus, quadricepsfemoris, hamstrings, and foot extensors and flexors.

Pulmonary function tests will be performed as a measure of the strengthof the muscles of the thoracic cage. Spirometry, with or withoutbronchodilators, will be employed to assess forced vital capacity (FVC)and forced expiratory volume in 1 sec (FEV1). In addition, maximalinspiratory and expiratory pressures (MIP and MEP) and maximum voluntaryventilation (MVV) will be recorded using standard techniques. The bestresult of three trials will be recorded. Lung volumes and diffusioncapacity will be assessed at baseline and repeated if clinicallyindicated.

Self-report assessments that capture global clinical improvement willinclude the Human Activity Profile (Fix, A. J., and Daughton, D. M.(1998) Human Activity Profile Professional Manual. PsychologicalAssessment Resources Inc.) and the SF-36v2 quality of lifequestionnaires (Ware et al. Med Care 30, 473-483 (1992)), to becompleted by the patients at the beginning and end of each crossoverperiod.

After baseline testing, patients will be randomized and ManNAc/placebotreatment will begin. The dose will be increased gradually every twodays, as follows: 1 capsule (500 mg) ManNAc/placebo four times a day(q.i.d.) for two days, followed by 1 capsule q.i.d. incremental increaseevery two days until the full dose of ˜10 g, or 5 capsules q.i.d. isreached (FIG. 7). Serum trough and peak levels (at 30 min, 1, 2 and 4hours post administration) of ManNAc, as well as glucose will beobtained after the first administration of ManNAc on the second day of acertain dose during the incremental dosing week. At the end of eachtreatment period trough and peak (1 hour) levels will be obtained aftereach dose over 24 hours.

Once the full dose of 10 g/day is reached, a repeat ECG or 24hambulatory ECG monitoring (Holter), spot urinalysis and the followingblood tests will be performed for safety reasons:

Total Volume # Drawn ml/Test (ml) CBC, ESR, platelets 1 3.0 3.0 Chem 20panel, CPK, 1 3.5 3.5 & fasting glucose, lipids PT, PTT 1 4.5 4.5 Total11.0The patients will be discharged home on the 10 g/day dose, to return forfollow-up admissions at one month and 3 months. This schedule will berepeated for the second crossover period after a 6-week washout period.

Follow-Up Studies

Follow-up evaluations will occur at the one-month time point for eachcrossover period, and at the end of each crossover period. They willconsist of a one-week admission involving repeat 24-hour urine studiesand the following blood tests:

Total Volume # Drawn ml/Test (ml) CBC, ESR, platelets 1 3.0 3.0 Chem 20panel, CPK, 1 3.5 3.5 & fasting glucose, lipids PT, PTT 1 4.5 4.5 HbA1C,Insulin 1 3.5 3.5 Free T4/TSH 1 3.5 3.5 FSH, LH, Test., Estradiol 1 5.05.0 Platelet pellet 1 5.0 5.0 Leucocyte pellet/amino acids 1 5.0 5.0Serum ManNAc, SA 1 4.0 8.0 Total 37.0

Follow-up procedures and consultations will involve repeat ECG or 24hambulatory ECG monitoring (Holter) and echocardiogram, musclequantitative strength assessments, functional muscle studies, PFTs, andfunctional and quality of life questionnaires.

To monitor treatment at home, patients will be asked to complete aweekly diary (available both electronically and in hard-copy) in orderto record missed doses, GI upset or other side-effects, and observedimprovements.

Statistical Considerations

Patients will be randomized using a permuted block size to either ManNAcor placebo (1:1) in a double-blind fashion (block randomization by theNational Institutes of Health Clinical Center Pharmacy) to ensurebalanced assignment to the two groups of patients with respect todisease duration and severity. There will be two 3-month crossoverperiods separated by at least 6-week washout period. Each patient willserve as his/her own control. The randomization code will not be brokenuntil completion of the study and analysis of the results.

The primary clinical outcome parameter will be change in quadricepsmuscle strength, assessed by maximum voluntary isometric contractiontesting (MVIC) that will be performed at baseline, 1 month afterinitiation of treatment and at the end of each treatment period. Changein strength (kg) will be expressed as % of baseline. Paired t-tests andthe Wilcoxon matched pairs signed rank test will be employed for theanalysis.

Secondary outcome parameters will include functional muscle testingusing the 6-minute walk test, functional reach, timed up-and-go, gripstrength and pulmonary function tests. Lastly, each patient's globalassessment of improvement will be based on the Human Activity Profile(ALSFRS) and SF-36 quality of life questionnaires, and specificself-assessment scores will be obtained for depression, fatigue, andfunction.

The estimated sample size is based upon different pieces of data,including the inventors' experience with four HIBM patients whose musclestrength was quantified before and after a month of intravenous immuneglobulin treatments as a source of sialic acid (Sparks, S., et al. BMCNeurol 7, 3 (2007)). There was a highly significant correlation betweenthe change in left quadriceps strength and the change in rightquadriceps strength. Therefore, the mean of the two sides as the primaryoutcome parameter will be used in larger clinical studies. Next, achange of 0% in patients before and after the 3-month placebo treatmentwill be assumed, based upon the fact that significant progression ofmuscle weakness takes years to occur. An estimated 10% standarddeviation is expected for the baseline and post-treatment measurements,and a 10% standard deviation for the mean difference, which would be 0for placebo treatment. The coefficient of variation for the MIVC methodof quantitative muscle strength testing in the literature is 6-15%.Andres, P. L., et al. Neurology 36, 937-941 (1986); Colombo, R., et al.Med Eng Phys 22, 167-174 (2000); A comparison of muscle strength testingtechniques in amyotrophic lateral sclerosis. Neurology 61, 1503-1507(2003); Mayhew, J. E., et al. Muscle Nerve 35, 36-42 (2007); Symons, T.B., et al. J Gerontol A Biol Sci Med Sci 60, 114-11 (2005).

A 20% improvement in quadriceps muscle strength is predicted, based uponseveral considerations. First, this would be a clinically significantimprovement, and a smaller benefit might not detectable or significant.Second, in intravenous immune globulin (IVIG) study conducted by theinventors, the mean (SD) improvement in strength for the 8 quadriceps of4 treated patients was 39±50%, so that a 20% improvement isconservative. Third, assuming complete absorption and conversion ofManNAc to sialic acid, 1000 times the sialic acid is delivered comparedto the amount of IVIG delivered in the previous study. A standarddeviation of muscle strength change under ManNAc treatment is estimatedto be 26%, because the SD/mean ratio of 26/20 is the same as the SD/meanratio of 50/39 observed in the IVIG study.

Under these conditions, a 20% difference will be detected using 18patients with a power of 0.90 and p=0.05. Twenty patients will betreated, with the expectation that two to drop out. Up to 30 patientswill hopefully be enrolled to obtain 20 who meet eligibilityrequirements.

This power analysis is conservative, since one-month data couldstrengthen the effect, a 20% improvement may be an underestimate, andthe relative SD for % improvement of 18 patients is likely to be lessthan that of the 4 patients upon whom we based the estimate. Changes insecondary outcome parameters will be analyzed using a one-tailed t-testand p=0.025.

Oral ManNAc supplementation could provide HIBM patients with transientimprovements in muscle strength and feelings of well-being. It may alsoprevent deterioration in their clinical course and be useful in treatingthe muscle weakness of HIBM. The clinical trial will also establish thesafety and tolerability of ManNAc for human use. This may serve as abasis for other potential therapeutic applications of ManNAc, such asthe potential benefit for podocytopathies and glomerular basementmembrane diseases suggested from our animal studies.

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All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby incorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims. As used herein and inthe appended claims, the singular forms “a,” “an,” and “the” includeplural reference unless the context clearly dictates otherwise. Thus,for example, a reference to “an antibody” includes a plurality (forexample, a solution of antibodies or a series of antibody preparations)of such antibodies, and so forth. Under no circumstances may the patentbe interpreted to be limited to the specific examples or embodiments ormethods specifically disclosed herein. Under no circumstances may thepatent be interpreted to be limited by any statement made by anyExaminer or any other official or employee of the Patent and TrademarkOffice unless such statement is specifically and without qualificationor reservation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

What is claimed:
 1. A method of treating a condition or disease in amammal in need thereof comprising administering an effective amount of asialic acid precursor, N-acetyl mannosamine or a derivative thereof, tothe mammal, wherein the derivative consists of Formula I:

wherein R₁, R₃, R₄, or R₅ is hydrogen, lower alkanoyl, carboxylate orlower alkyl; R₂ is lower alkyl, lower alkanoylalkyl, lower alkylalkanoyloxy; and the condition or disease is muscular atrophy, musculardystrophy, high blood pressure, or diabetes.
 2. The method of claim 1,comprising administering a therapeutic amount of N-acetyl mannosamine tothe mammal.
 3. The method of claim 2, wherein the therapeutic amount isabout 0.1 g to about 50 g N-acetyl mannosamine per day.
 4. The method ofclaim 2, wherein the therapeutic amount is administered in a unit dosageof about 0.01 g to about 50 g N-acetyl mannosamine per unit dosage. 5.The method of claim 1, comprising sustained release of the sialic acidprecursor, N-acetyl mannosamine or a derivative thereof.
 6. The methodof claim 1, comprising topical administration of the sialic acidprecursor, N-acetyl mannosamine or a derivative thereof.
 7. The methodof claim 1, wherein them method reduces hematuria in the mammal.
 8. Amethod of reducing hematuria in a mammal in need thereof comprisingselecting a mammal with a condition or disease, and administering to themammal an effective amount of a sialic acid precursor, N-acetylmannosamine or a derivative thereof, to the mammal, wherein thederivative consists of Formula I:

wherein R₁, R₃, R₄, or R₅ is hydrogen, lower alkanoyl, carboxylate orlower alkyl; R₂ is lower alkyl, lower alkanoylalkyl, lower alkylalkanoyloxy; and wherein the condition or disease is hereditaryinclusion body myopathy, muscular atrophy, muscular dystrophy, highblood pressure, diabetes, or diabetic nephropathy.
 9. The method ofclaim 8, comprising administering a therapeutic amount of N-acetylmannosamine to the mammal.
 10. The method of claim 9, wherein thetherapeutic amount is about 0.1 g to about 50 g N-acetyl mannosamine perday.
 11. The method of claim 9, wherein the therapeutic amount isadministered in a unit dosage of about 0.01 g to about 50 g N-acetylmannosamine per unit dosage.
 12. (The method of claim 8, comprisingsustained release of the sialic acid precursor, N-acetyl mannosamine ora derivative thereof.
 13. The method of claim 8, comprising topicaladministration of the sialic acid precursor, N-acetyl mannosamine or aderivative thereof.
 14. The method of claim 9, wherein the N-acetylmannosamine is administered orally to the mammal.
 15. The method ofclaim 9, wherein the N-acetyl mannosamine is microencapsulated.
 16. Themethod of claim 8, wherein the method also decreases proteinuria in themammal.